Combinatorial Selection, Inhibition, and Antiviral Activity of DNA Thioaptamers Targeting the RNase H Domain of HIV-1 Reverse Transcriptase

Abstract

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K(d)) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi <or= 0.005.

Nucleic Acid Aptamers Based on the G-Quadruplex Structure: Therapeutic and Diagnostic Potential

“…Several sets of DNA-based aptamers were described, a number of which exhibited high G contents, hence they were amenable to G-4 folding [63][64]. In particular, very recent work reinvestigated several single-stranded DNA aptamers reported to bind HIV-1 RT selectively [65].…”

Gene therapy progress and prospects: RNA aptamers

“…36 As intracellularly expressed antiHIV-1 RT aptamers had shown promise as antiviral agents in cell culture assays, additional RT-directed aptamers continue to be described, including those that selectively inhibit the RNaseH domain of HIV-1 RT. 45,46 One of the more exciting antiHIV-1 aptamers reported to date is the RNA aptamer B4 that is specific for gp120 of HIV-1 strain R5. The envelope protein gp120 controls viral entry through its interaction with chemokine receptors.…”

Selection of a novel DNA thioaptamer against HER2 structure

“…Among these modification methods, phosphate backbone modification with phosphorothioate is a promising strategy, since it does not hamper the target-binding affinity of the oligonucleotides. However, complete substitution of all phosphoryl oxygen in DNA with sulfur appears to make the thioaptamers too sticky, such that they lose their ability to discriminate between target and non-target proteins [33,34]. A solution of this issue is to replace only one of the four bases in DNA with phosphorothioate to generate thioaptamers [14].…”